Hematopoietic stem cell (HSC) transplantation is increasingly used for the treatment of a number of malignant and non-malignant disorders of both hematopoietic and non-hematopoietic origin. However, rejection responses mediated by the immune system of the donor against the recipient, termed graft versus host disease (GvHD) remains a major cause of morbility. Organ transplantation is the best available established technique for the treatment of end stage failure of most essential organs (liver, heart, and lungs), but allograft rejection mediated by the host is a major hurdle to long-term graft survival. A panel of immunosuppressive drugs is now available to prevent acute GvHD and allograft rejection including steroids, cyclosporin, metotrexate, cyclophosphamide, anti-thymocyte globulin, and anti-CD3 mAb. While these agents have significantly improved graft outcomes, their use have been associated with numerous and rather significant toxicities. Moreover, continuous drug administration leads to a sustained state of immunosuppression with consequent high risk of infections. All these effects are linked to the non-selective mode of action of the immunosuppressive drugs.
A valid alternative to immunosuppressive regimens for prevention of GvHD and of allograft rejection is the induction of tolerance to the alloantigens expressed by the recipient or the graft. This tolerance strategy should selectively target only a small fraction of potentially alloreactive T cells and leave the rest of the immune system intact.
In autoimmune diseases, undesired immune response to self-antigens lead to destruction of peripheral tissues. Treatments of autoimmune diseases are currently based on modulation of inflammation and non-specific immunosuppression. Similarly to the prevention of allograft rejection and GvHD, this approach is frequently not effective long-term due to the side effects of immunosuppression including infections and cancer, and high risk of disease relapse once the drug is withdrawn. An alternative strategy is based on the induction of specific immune tolerance with the ultimate goal to down-regulate the pathogenic immune response to self-antigens and to keep intact the mechanisms of host defense.
In chronic inflammatory diseases and in allergies an altered immune response to pathogenic and non-pathogenic antigens occurs. This may be due to an unbalance between effector and regulatory immune responses. Conventional anti-inflammatory or immunosuppressive therapies are insufficient to restore this balance. Moreover, the benefit of these therapies is not long-lasting after drugs withdrawn. The induction of antigen-specific tolerance mechanisms able to suppress undesired responses would represent a major advantage. Indeed, IL-10-producing T cells with regulatory properties, which are specific for different non-pathogenic antigens have been isolated in healthy donors.
In addition to central tolerance which occurs during T-cell ontogeny in the thymus and is mediated by clonal deletion of self-reactive T cells, peripheral T-cell tolerance is operational throughout life and is designed to control responses towards self antigens and foreign antigens which are not harmful. Peripheral T-cell tolerance can be induced and maintained by a variety of mechanisms, including deletion, induction of T-cell hypo-responsiveness, and differentiation of T regulatory (Tr) cells. Tr cells include a wide variety of cells with a unique capacity to inhibit effector T-cell responses. Although T cells with suppressive activity exist in all T-cell subsets, the best characterized are comprised in the CD4+ T population. The two most relevant classes of Tr cells described within the CD4+ subset to date are: T regulatory type 1 (Tr1) cells (1) and CD4+CD25+ Tr cells (2). These two Tr cell subsets differ in a number of important biological features, including their specific cytokine secretion profile, cellular markers, ability to differentiate in response to Ag specific stimuli, and dependency on cytokines versus cell-cell contact mechanisms for mediating suppressive activity.
IL-10 and Type 1 T Regulatory (Tr1) Cells.
IL-10 plays a central role in controlling inflammatory processes, suppressing T cell responses, and maintaining immunological tolerance (reviewed in (3)). IL-10 inhibits IFN-γ and IL-2 production by T cells (4). It has anti-inflammatory effects inhibiting production of pro-inflammatory cytokines, such as TNF-α, IL-1, and IL-6, and chemokines, such as IL-8 and MIP1α, produced by activated antigen-presenting cells (APC), neutrophils, eosinophils, and mast cells. Furthermore, IL-10 down-regulates the expression of MHC class II, costimulatory, and adhesion molecules (5-7) on APC, and modulates their stimulatory capacity (8). Importantly, IL-10 is crucial for the differentiation of adaptive type 1 T regulatory (Tr1) cells (1). Tr1 cells are characterized by a unique cytokine secretion profile, upon TCR activation they secrete high levels of IL-10, significant amounts of IL-5, TGF-β and low levels of IFN-γ, and IL-2 but not IL-4 (1). Ag-specific murine Tr1 cells can be indeed differentiated in vitro by repetitive TCR stimulation in the presence of high doses of IL-10 (1). Furthermore, treatment of mixed lymphocyte reaction (MLR) cultures with IL-10 (9) (and TGF-β in the mouse (10)) results in T-cell anergy. Importantly, allo-reactive Tr1 cell clones from healthy individuals have been originally isolated by limiting dilution of in vitro IL-10-anergized CD4+ T cells (1).
The first suggestion that human Tr1 cells are involved in maintaining peripheral tolerance in vivo came from studies in severe combined immunodeficient (SCID) patients successfully transplanted with HLA-mismatched allogenic stem cells. In the absence of immunosuppressive therapy, these patients do not develop GvHD. Interestingly, high levels of IL-10 are detected in the plasma of these patients and a significant proportion of donor-derived T cells, which are specific for the host HLA antigens and produce high levels of IL-10, can be isolated in vitro (11). Importantly, IL-10-anergized cells preserve their ability to proliferate in response to nominal antigens, such as Tetanus Toxoid and Candida Albicans, indicating that IL-10 induces an Ag-specific anergy (Bacchetta unpublished data). In a preclinical model of bone marrow transplantation, transfer of donor CD4+ T cells anergized ex-vivo by host APC in the presence of IL-10 and TGF-β results in a markedly decreased GvHD in MHC class II mismatched recipients (10, 12). These data offer a strong rationale for the development of a clinical protocol using co-transfer of ex-vivo IL-10-anergized cells of donor origin in patients undergoing haplo-identical HSC transplantation.
Tolerogenic Dendritic Cells (DC)
DC are highly specialized APC that classically initiate Ag-specific immune responses upon infection (13). This process involves the terminal maturation of DC, typically induced by agents associated with microbial infection. It is now clear that DC can be not only immunogenic but also tolerogenic. In steady state DC remains immature DC and can induce tolerance via deletion of Ag-specific effector T cells and/or differentiation of Tr cells (14-18). Repetitive stimulation of naïve cord blood CD4+ T cells with allogeneic immature DC results in the differentiation of IL-10-producing Tr cells (19), which suppress T-cell responses via a cell-contact dependent mechanism. The authors recently reported that peripheral blood naïve CD4+ T cells stimulated with allogeneic immature DC become increasingly hypo-responsive to re-activation with mature DC and after three rounds of stimulation with immature DC, they are profoundly anergic and acquire regulatory function. These T cells are phenotypically and functionally similar to Tr1 cells since they secrete high levels of IL-10 and TGF-β, suppress T-cell responses via an IL-10- and TGF-β-dependent mechanism, and their induction can be blocked by anti-IL10 mAb (20). Not only immature DC but also specialized subsets of tolerogenic DC can drive the differentiation of Tr cells. Maturation and function of DC can be regulated at different levels (21). Both pharmacological and biological agents have been shown capable of inducing tolerogenic DC (22). Several biological agents including IL-10 (23, 24), TGF-β (25), IFN-α (26, 27), and TNF-α (28) can induce Tr cells. The presence of IL-10 during maturation of DC generate tolerogenic DC (23, 24), which express low levels of costimulatory molecules and MHC class II (24), display low stimulatory capacity (3, 29), and induce antigen-specific T cells anergy in both CD4+ and CD8+ T cells (23, 24).
It has been already described that IL-10 during DC differentiation results in a population of macrophage-like cells with low stimulatory capacity but mature phenotype (8, 30). Herein, we demonstrated that IL-10 treatment induces the differentiation of a unique subset of DC (Tr-DC) characterized by the expression of CD14, CD11c, CD11b, CD83, CD80, CD86, CD71 and HLA-DR, but not CD1a. Tr-DC express immunoglobulin-like transcript (ILT-) 2, ILT-3, ILT-4, and the non classical MCH class I molecule HLA-G. Tr-DC secrete significantly higher levels of IL-10 compared to immature DC, whereas the amounts of IL-12 are comparable to those produced by immature DC. Interestingly, IL-10/IL-12 ratio is maintained upon activation with LPS and IFN-γ. Tr-DC display lower stimulatory capacity compared to immature DC, and, importantly, induce Tr1 cells. Thus, IL-10 promote the differentiation of a new subset of tolerogenic DC which can be used to generate anergic Tr1 cells with limited in vitro manipulation and suitable for potential clinical use to restore peripheral tolerance.
Induction of T cell anergy by IL-10-treated DC has been suggested by Zheng et al. (2004). The authors have generated immature DC by culture of adherent cells with IL-4 and GM-CSF treatment. The immature DC obtained after 7 days are then washed and cultured with IL-10 for additional 2 days. The resulting IL-10-treated immature DC present a phenotype very different from the one of the Tr-DC obtained in the present invention. Indeed, the cells obtained in Zheng et al. are CD83 negative, CD86 low and HLA-DR low.
The protocol proposed by Levings et al. (2005) leads to the induction of Tr1 cells by repetitively stimulation of CD4+ T cells using immature DC, which are different from the Tr-DC generated in the present invention.
The international patent application WO2004/087899 discloses a method for obtaining Tr1 cells from T cells by means of specialized DC. DC are obtained from CD34+ cells in presence of IL-4, GM-CSF and IL-10. However, by contrast with the Tr1 DC of the present invention, the resulting DC express low level of CD11c, HLA-DR, CD80 and CD86, and are CD14 negative.
The international patent application WO03/000199 provides compositions which comprise at least two of a CD4+CD25+ T cell, IL-10, a CD8+CD28− cell and a vitamin D3 analog. This application also discloses a method for generating a tolerogenic antigen-presenting cell, which comprises contacting the cell with an effective amount of IL-10, a CD4+CD25+ T cell and/or a vitamin D3 analog. A method for increasing the expression of ILT3 and/or ILT4 by an antigen-presenting cell which comprises contacting the cell with an effective amount of IL-10, a CD4+CD25+ cell and/or a vitamin D3 analog and methods for inhibiting the onset of or treating the rejection of an antigenic substance and inhibiting the onset of or treating an autoimmune disease in a subject are provided.
The U.S. Pat. No. 6,277,635 describes IL-10 for producing a population of cells which are capable of inhibiting or suppressing reactions to alloantigens, for example in graft-versus-host disease or tissue rejection. IL-10 for reducing responses in mixed lymphocyte response (MLR) is also described. Exogenous or induced endogenous IL-10 may be used for the inhibition or suppression of the reactions to alloantigens. The Tr-DC method of the present invention differs from the IL-10 protocol to anergize T cells in vitro as follow:                Anergy by Tr-DC can be induced in all the individuals.        Anergic T cells induced by Tr-DC are more stable compared to those obtained with IL-10.        T-cell cultures obtained with Tr-DC display higher cell recovery compared to those obtained with IL-10.        IL-10 and Tr-DC are comparable in inducing T-cell anergy in haplo-identical pairs. Importantly, in haplo-identical pairs in which IL-10 does not induce anergy, Tr-DC do.        In HLA-matched un-related (MUD) pairs the use of DC is required to stimulate host-specific T-cell responses, therefore Tr-DC are necessary for T-cell anergy induction.        Lower number of cells from both recipient and donor are required for the in vitro manipulation to generate anergized T cells with the Tr-DC of the present invention.        
The United States patent application 20070009497 relates to culture-expanded T suppressor cells and their use in modulating immune responses. This invention provides methods of producing culture-expanded T suppressor cells, which are antigen specific, and their use in modulating complex autoimmune diseases. In particular a method for producing an isolated, culture-expanded T suppressor cell population, comprising: (a) contacting CD25+CD4+ T cells with DC and an antigenic peptide, an antigenic protein, or a derivative thereof, or an agent that cross-links a T cell receptor on said T cells in a culture, for a period of time resulting in antigen-specific CD25+CD4+ T cell expansion; and (b) isolating the expanded CD25+CD4+ T cells obtained in (a), thereby producing an isolated, culture-expanded T suppressor cell population is provided. The DC population describes in this application display very different characteristics than the Tr-DC population of the present invention.
The International patent application WO03102162 relates to tolerogenic DC and methods for enriching for these cells in tissue preparations and using the cells for preventing or minimizing transplant rejection or for treating or preventing an autoimmune disease. A human tolerogenic DC having surface antigens DEC205 and B220, but not CD19 is described.
HLA-G and Immunomodulatory Properties
HLA-G, a non-classical MHC class I molecules, is a low polymorphic molecule. Compared with the classical class I genes, the most polymorphic genes in the human genome, HLA-G has relatively little polymorphism in its coding region (31). The HLA-G gene has eight exons encoding a signal peptide (exon 1), the α1, α2, and α3 domains (exons 2, 3, and 4, respectively), the transmembrane domain (exon 5), and the intracellular domain (exons 6 and 7), similar to other class I genes. However, a premature stop codon in exon 6 results in a truncated cytoplasmic tail that reveals a cryptic retrieval motif (32). This results in the slow turnover and prolonged expression of HLA-G at the cell surface. HLA-G encodes multiple isoforms as a result of alternative splicing. The full-length isoform HLA-G1 is structurally similar to other class I genes, except for the truncated cytoplasmic tail. The G2 isoform results from the removal of exon 3 and homodimerizes to form an HLA class II-like structure (33). HLA-G1 and HLA-G2 isoforms can be also expressed as soluble proteins (HLA-G5 and -G6, respectively) due to the inclusion of intron 4 sequences in the mature mRNA, resulting in secreted proteins with an additional 21 amino acids (encoded by intron 4 sequences) following the α3 domain (34). HLA-G3 results from the removal of exons 3 and 4. Additional isoforms are HLA-G4 and -G7.
HLA-G has been extensively studied in pregnancy and it is known to be the major contributor to induction and maintenance of foetal-maternal tolerance (31, 35). HLA-G inhibits cytolytic activities of both NK and CTL (36), and allo-specific T-cell proliferation (37, 38). A positive correlation between allograft acceptance and HLA-G expression on both graft cells (39, 40) and T cells (38) has been reported (41), indicating a role of HLA-G in modulating allo-responses. In addition, HLA-G acts as a negative regulator of tumor immune responses through several mechanisms including, inhibition of angiogenesis, prevention of antigen recognition and T-cell migration, and suppression of T and NK cytolytic effects (42). Antigen-presenting cells expressing HLA-G1 are poor stimulators and are able to promote the induction of anergic/suppressor CD4+ T cells (43). Moreover, HLA-G binds to the inhibitory molecules immunoglobulin-like transcript (ILT)-2 and ILT-4 expressed on DC (39, 44). It has been shown that engagement of ILT-4 by HLA-G prevents the up-regulation of costimulatory molecules, inhibits DC maturation (45), and promotes the differentiation of anergic/suppressor CD4+ T cells (46). The authors demonstrated that soluble HLA-G alone or in combination with IL-10 promotes the differentiation of a population of CD4+ T cells with low proliferative capacity and suppressor functions. Soluble HLA-G-induced Tr cells produce TGF-β, intermediate levels of IL-10 and IFN-γ, but low levels of IL-2, and IL-4, express high levels of granzyme B, CTLA4, CD25, but not FOXP3. Thus soluble HLA-G is a new immunomodulatory compound able to promote the differentiation of a population of CD4+ T cells with regulatory activity.